Enzyme-prodrug therapy for prosthetic joint repair

ABSTRACT

The invention relates to the use of gene therapy in the treatment of aseptic loosening of orthopaedic prostheses and discloses methods of refixing such prostheses without open revision surgery. In particular, it provides adenoviral vectors and prodrugs for simultaneous, separate or sequential use in the destruction of interface tissue allowing subsequent recementing of loose prostheses in a minimally invasive manner.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.11/072,980, filed Mar. 4, 2005, which claims priority under 35 U.S.C.§119(e) to Provisional Application Ser. No. 60/563,067, Filed: Apr. 16,2004 and Provisional Application Ser. No. 60/613,305, Filed: Sep. 27,2004 and claims priority under 35 U.S.C. §119(a) to Great Britain PatentApplication No. 0405103.3, Filed: 6 Mar. 2004 and Great Britain PatentApplication No. 0420217.2, Filed: 11 Sep. 2004. All applications arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the use of gene therapy in the treatment ofaseptic loosening of orthopaedic prostheses. In particular, it disclosesmethods of refixing such prostheses without open revision surgery.

BACKGROUND TO THE INVENTION

Approximately 1 million total hip replacement (total hip arthroplasty)operations are carried out world-wide annually, with more than 120,000of these undertaken in the USA, and about 35,000 in England alone (NIHConsensus Statement, 1994; NHS Review 1996). This is likely to increaseto approximately 3 million worldwide per annum within the next decade.Hip replacements are very often performed in elderly patients and,amongst this group, loosening of one or both components of theprosthesis, resulting in severe mobility restriction, occurs within 15years in about a third of patients. Where prosthetic loosening occurs,patients' experience increased pain and walking difficulty and have ahigher risk of dislocations and pathological fractures. Within 10 years,approximately 10% of all patients require revision surgery, which has ahigh rate of complications and failure (Hellman et al, 1999).

The most common cause of implant failure is aseptic loosening as aresult of particulate-induced osteolysis. Wear particles, such asparticles of polyethylene, polymethylmethacrylate, titanium, cobaltchrome or ceramic debris, depending on the type of prosthesis, stimulatean inflammatory response termed periprosthetic osteolysis (Goldring etal, 1986). The phagocytosis of wear particles by macrophages activatesthem, leading to secretion of the inflammatory cytokines IL-1, TNF-α,and IL-6. The resulting chronic inflammatory response eventuallyproduces a pseudomembrane of granulomatous ‘interface tissue’ includingactivated macrophages, fibroblasts, giant cells and osteoclasts, similarto the pannus characteristic of arthritic joints. The end result of thiscomplex inflammatory and proliferative foreign body response isosteoclast-mediated resorption of bone, leading to loosening of one orboth components of the prosthetic implant. Prostheses for total hiparthroplasty consist of two components. An artificial socket, oracetabular component, is located in a prepared cavity in the acetabulumof the pelvis. This articulates with a femoral component comprising aball attached to a process, which is introduced into a prepared cavityin the medulla of the femur. Many variations of both components exist,and they may be retained with or without cements.

Aseptic loosening eventually leads to an unacceptable degree of pain,immobility or walking difficulties and instability, with a higher riskof dislocations and pathological fractures. In some patients revisionsurgery may be undertaken to remove the inflammatory tissue and replacethe prosthesis. However, revision surgery is very expensive and has ahigh morbidity and mortality rate, especially in elderly patients (whoare in the majority). In patients with cardiac insufficiency revisionsurgery often has major complications such as myocardial failure orcoronary artery disease (Strehle et al, 2000). Many patients are noteligible for revision surgery because the risk of mortality isconsidered to be too high. There is no alternative treatment for suchpatients, who are then wheelchair-bound. The clinical need for a lesstraumatic alternative to revision surgery for treatment of loosenedprostheses is therefore clear. At present experimental approaches tothis problem are preventative rather than therapeutic. One suchpreventative approach to controlling aseptic loosening involves the useof bisphosphonate compounds, especially alendronate, as either asystemic medication or as a component of a cement used to fix suchprostheses (U.S. Pat. No. 5,972,913, WO 96/39107, Shanbhag et al, 1997,Leung et al, 1999). However, although bisphosphonates are known toproduce an increase in skeletal bone density, they have not been shownto have a significant effect in treating rheumatoid arthritis, whichshares many similar pathological features with periprostheticosteolysis, nor on periprosthetic osteolysis itself (Ralston et al,1989; Eggelmeijer et al, 1996; Ulrich-Vinther, 2002). It thus remains tobe seen whether bisphosphonates have a useful role to play in theprevention of aseptic loosening.

In an attempt to prevent osteoclast-mediated periprosthetic boneresorption directly, an alternative preventative approach involves genetherapy (reviewed in Wooley and Schwarz, 2004), using an osteoclastinhibitory protein, osteoprotegerin, delivered by means ofadeno-associated virus vector has been described (Ulrich-Vinther, 2002).Osteoprotegerin is a competitive inhibitor of an osteoclastdifferentiation factor, receptor activator of nuclear factor κB ligand(RANKL), which binds to a receptor expressed on the surface ofmacrophage-derived osteoclast precursor cells, known as receptoractivator of nuclear factor κB (RANK). RANKL is secreted by osteoblasts,stromal cells and activated T cells at an early stage of theinflammatory response initiated by macrophage phagocytosis of wearparticles (Teitelbaum, 2000). Binding of RANKL to RANK leads toactivation of osteoclast precursor cells, differentiation, andstimulation of bone resorption. Binding of RANK by osteoprotegerin failsto activate the osteoclast precursor cells with the result thatosteoprotegerin competitively inhibits RANKL.

Ulrich-Vinther et al used a recombinant adeno-associated virus (rAAV)vector to express osteoprotegerin and inhibit titanium particle-inducedresorption in a mouse calvarial resorption model. Titanium particleswere implanted on the calvaria (bones of the vault of the skull) and thevector administered by intramuscular injection into the quadriceps. Theinhibitory effect of the osteoprotegerin was therefore systemic, withdetectable increases in serum levels, and this appeared to be successfulin inhibiting the experimental titanium-induced osteoclastogenesis andbone resorption seen in the untreated controls. Although interesting, itremains to be seen whether this model will form the basis of a viablepreventative for clinical periprosthetic osteolysis. Even if effective,it is unclear what long-term systemic effects prolonged elevations inserum osteoprotegerin levels might have. For example, such a strategywould need to demonstrate a lack of deleterious effects on normalosteoclast function in bone remodelling.

There remains a need for effective treatments for the common anddebilitating condition of periprosthetic osteolysis and its resultantaseptic loosening.

One approach to preferentially killing pathological cells, most widelyused for treating cancer, is to introduce a gene into the target cellsthat encodes an enzyme capable of converting a prodrug of relatively lowtoxicity into a potent cytotoxic drug. Systemic administration of theprodrug is then tolerated since it is only converted into the toxicderivative locally, for example in a tumour, by cells expressing theprodrug-converting enzyme. This approach is known as gene-directedenzyme prodrug therapy (GDEPT), or when the gene is delivered by meansof a recombinant viral vector, virus-directed prodrug therapy (VDEPT)(McNeish et al, 1997).

An example of an enzyme/prodrug system is nitroreductase and theaziridinyl prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) (Knoxet a/1988). Following the observation that the Walker rat carcinoma cellline was particularly sensitive to CB1954, it was shown that this wasdue to the expression of the rat nitroreductase DT diaphorase. However,since CB 1954 is a poor substrate for the human form of this enzyme,human tumour cells are far less sensitive to CB1954. GDEPT was conceivedas a way of introducing a suitable nitroreductase, preferably withgreater activity against CB1954, in order to sensitise targeted cells.The Escherichia coli nitroreductase (EC1.6.99.7, alternatively known asthe oxygen-insensitive NAD(P)H nitroreductase or dihydropteridinereductase, and often abbreviated to NTR) encoded by the NFSB gene(alternatively known as NFNB, NFSI, or DPRA) has been widely used forthis purpose (Reviewed in Grove et al, 1999). The NFSB-encodednitroreductase (NTR) is a homodimer that binds two flavin mononucleotide(FMN) cofactor molecules. Using NADH or NADPH as an electron donor, andbound FMN as a reduced intermediate, NTR reduces one or other of the twonitro-groups of CB 1954 to give either the highly toxic 4-hydroxylaminederivative or the relatively non-toxic 2-hydroxylamine. Within cells,5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, probably via afurther toxic metabolite, becomes very genotoxic (Knox et al, 1991). Theexact nature of the lesion caused is unclear, but is unlike that causedby other agents. A particularly high rate of inter-strand cross-linkingoccurs and the lesions seem to be poorly repaired, with the result thatCB 1954 is an exceptionally affective anti-tumour agent (Friedlos et al,1992).

The aim of GDEPT is to obtain efficient conversion of a prodrug such asCB1954 in target cells in order to kill not only NTR-expressing cellsbut also bystander tumour cells that may not have been successfullytransfected or transduced.

Another enzyme-prodrug system used in this way is that of a cytochromeP450 as a prodrug-converting enzyme and acetaminophen as the prodrug, asdescribed in international application WO 00/40271 (incorporated hereinin its entirety). A number of cytochrome P450 enzymes, naturallyexpressed in the liver (for example CYP1A2, CYP 2E1 and CYP3A4) arecapable of converting acetaminophen into a highly cytotoxic metabolite,N-acetylbenzoquinoneimine (NABQI). This system has been proposed for avariety of clinical applications, especially in the field of cancertherapy. Cytochrome P450 enzymes are also capable of activating severalconventional cytotoxic prodrugs, for example cyclophosphamide andifosfamide (Chen and Waxman, 2002).

A number of other enzyme-prodrug systems are widely used, including HSVthymidine kinase and ganciclovir (Moolten, 1986), cytosine deaminase and5-fluorocytosine (Mullen et al, 1992).

Goossens et al (1999) describe a viral gene therapy approach to infectand kill isolated cultured synovial cells in vitro, and to kill pannustissue in a monkey collagen-induced arthritis model in which inflamedjoints are induced by collagen injections. Inflamed joints in suchanimals contain a hyperplastic tissue resulting from the chronicinflammation termed pannus.

SUMMARY OF THE INVENTION

As used herein:

“Cell-type selective” means; facilitating expression preferentially in alimited range of tissues. Preferably, such expression is substantiallylimited to a single tissue or cell type.

An “operably-linked promoter” is one in a substantially adjacentcis-relationship, wherein said promoter directs expression of theoperably-linked element.

“Periprosthetic” relates to the space surrounding any part of animplanted prosthesis

“Periprosthetic osteolysis” is synonymous with “aseptic loosening” andrelates to any progressive loosening of an implanted prosthesis notassociated with frank infection or trauma.

“Interface tissue” is synonymous with “osteolytic membrane” and meansinflammatory tissue in the periprosthetic space round an implantedprosthesis, implicated in periprosthetic osteolysis.

“Prosthesis” or “Orthopaedic implant” as herein used means any materialor device surgically implanted into a bony structure of an animal orhuman.

An aim of the invention is to provide a non-surgical alternative torevision surgery for treatment of loosened prostheses that destroysinterface tissue (and the cells within it that are involved in theinflammatory processes and bone resorption) and allows the implant to berecemented.

The invention seeks to achieve this by using an enzyme-prodrug therapystrategy using a gene therapy vector to deliver a prodrug-convertingenzyme to cells in the interface tissue, thus sensitising them to aparticular prodrug. Administration of the prodrug leads to itsconversion to an active cytotoxic drug in the target cells, killing theinterface tissue. Release of active cytotoxic drug from lysed interfacecells may also kill neighbouring interface or inflammatory cells(‘bystander’ killing), which is advantageous in that cells that haveescaped direct vector delivery (by transduction, for viral vectors, ortransfection for non-viral vectors) are also eliminated.

In one strategy, a viral vector carrying nucleic acid encoding theenzyme is injected into the intra-articular space, and the prodrugsubsequently administered through a small drill hole, which can also beused to inject cement to refix the prosthesis in situ. Alternatively,the prodrug may be administered by intra-articular injection.Arthrography has shown that the interface tissue forms a continuousclosed compartment around the loosened prosthesis, which allows a highlocal concentration of both vector and prodrug to be achieved with verylow risk of systemic escape. The concept thus offers more favourablecircumstances in terms of both efficacy and safety than intra-tumoralinjection in cancer patients, a procedure with which there isconsiderable clinical experience. In the case at least of adenoviralvectors, it may be preferable to remove existing fluid in theintra-articular/periprosthetic space before introducing the vectors, toreduce the possibility of neutralising antibodies in the fluidinactivating the vector and preventing satisfactory levels oftransduction.

Preferably, following introduction of the prodrug and consequent killingof cells of the interface tissue, said tissue is removed. This may beaided by the introduction of, either simultaneous with, or subsequentto, introduction of the prodrug, one or more enzymes capable ofdigesting extracellular components of the interface tissue, such ascollagenase, elastase or hyaluronidase, matrix metalloproteases orcathepsins.

Other compounds useful for this purpose include the chelating agentsEDTA (Ethylenediamine-N,N,N′,N′-tetra-acetic acid) and EGTA (Ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid). Such treatmentdigests and loosens the interface tissue, such that it may be flushedout through a suitable drill hole or via a wide bore needle introducedinto the intra-articular space.

The fully loosened and debrided implant is then recemented, to solidlyreattach all loosened components and restore a fully functionalprosthetic joint.

Alternatively, especially with prodrugs such as acetaminophen with verylow systemic toxicity, the vector encoding the prodrug converting enzyme(such as cytochrome P450) may be injected locally, so that only cellswithin the interface tissue/joint compartment are transduced, whilst theprodrug is subsequently administered systemically.

In one aspect of the invention, the approach is to kill cells residentin the interface tissue, irrespective of their type. In practice, thepredominant cells are fibroblasts responsible for producing theextracellular matrix proteins of which much of the tissue is comprised,and cells of the monocyte/macrophage lineage responsible forinflammatory effects. In this case, the expression of the enzyme encodedby the vector is controlled by a strong non-cell type specific promoter,providing high level expression in a variety of cell and tissue types,such as the cytomegalovirus early/immediate promoter and the cytotoxiceffect is limited to cells of the interface tissue by the physicalconstraints of the space into which the vector and/or prodrug areinjected. The normal cells of most concern from the safety viewpoint arethe osteoblasts responsible for bone regeneration. In most instances,and with most gene delivery vectors, these cells are inaccessible tovector injected into the periprosthetic space, hence are not transducedor transfected, do not express the prodrug converting enzyme even with anon-cell type specific promoter, and are therefore not killed uponsubsequent administration of the prodrug.

Examples of such non-cell specific promoters include: cytomegalovirusimmediate/early promoter, Rous sarcoma virus long terminal repeat (RSVLTR), murine leukaemia virus LTR, simian virus 40 (SV40) early or latepromoters, herpes simplex virus (HSV) thymidine kinase (tk) promoter,actin or ubiquitin promoters.

In some circumstances it may be advantageous to achieve more selectivecell killing, in which case the enzyme encoded by the vector may beexpressed under the control of a tissue- or cell type-selectivepromoter. Use of such a promoter permits selective killing of cells ofparticular lineages, such as fibroblasts, cells of themonocyte/macrophage lineage or, more specifically, cells of a particularphenotype, such as osteoclast precursor cells, or fully differentiatedosteoclasts.

Examples of promoters suitable for preferentially expressing a gene,such as a gene encoding a prodrug-converting enzyme, in cells of themonocyte/macrophage lineage include, c-fes and CD68. Promoterscharacterised by containing one or more binding sites for thetranscription factor PU.1 are generally suitable (Greaves and Gordon,2002).

Promoters suitable for expressing a gene preferentially in osteoclastsor osteoclast precursors include the tartrate-resistant acid phosphatase(TRAP) promoter, the RANK promoter and the cathepsin K promoter.Promoters characterised by containing one or more binding sites(E-boxes, containing the consensus binding sequences5′-CA(^(T)/_(G))GTG) for microphthalmia transcription factor family(MITF, TFE3, TFEB and TFEC), optionally also containing binding sitesfor the transcription factor PU.1 are generally suitable (Motyckova etal, 2001; Mansky et al, 2002, Greaves and Gordon, 2002).

By the use of such specific promoters, expression of the enzyme may berestricted to particular target cells, such as those responsible forlaying down of extracellular matrix proteins such as collagen(fibroblasts), those responsible for secreting inflammatory cytokines(such as macrophages) or those responsible directly for bone resorption(osteoclasts), whilst protecting other cell types (such as osteoblasts,responsible for depositing new bone).

The various possible combinations of local administration of vectorand/or prodrug with or without tissue-selective expression allownon-surgical treatment of loosened prostheses and recementation of theimplant, overcoming limitations in the prior art methods aimed atpreventing periprosthetic loosening by systemic administration ofcompounds such as bisphosphonates, or of systemic expression of highlybioactive molecules such as osteoprotegerin.

Accordingly, the invention provides an isolated polynucleotide encodingan enzyme capable of converting a prodrug into an active cytotoxiccompound, expression of the enzyme being controlled by anoperably-linked promoter that gives substantially cell type-selectiveexpression. Preferably expression is restricted to cells of themonocyte/macrophage lineage. Preferred examples such promoters includethe promoters of such genes as c-fes, and CD68. Promoters characterisedby containing one or more binding sites for the transcription factorPU.1 are generally suitable.

Alternatively, expression is restricted to fibroblasts.

More preferably expression is restricted to osteoclasts or osteoclastprecursors. Amongst suitable promoters providing such expression arethose naturally functionally linked to genes such as tartrate-resistantacid phosphatase (TRAP), receptor activator of nuclear factor κB (RANK)and cathepsin K. Promoters characterised by containing one or morebinding sites (E-boxes, containing the consensus binding sequences5′-CA(^(T)/_(G))GTG) for microphthalmia transcription factor family(MITF, TFE3, TFEB and TFEC), optionally also containing binding sitesfor the transcription factor PU.1 are generally suitable.

Preferably, the enzyme encoded is a nitroreductase, preferably anitroreductase suitable for the activation of the prodrug CB1954(5-(aziridin-1-yl)-2,4-dinitrobenzamide). Alternatively, it a cytochromeP450. Other suitable enzyme/prodrug systems include HSV thymidine kinaseand ganciclovir (Moolten, 1986), cytosine deaminase and 5-fluorocytosine(Mullen et al, 1992).

In another aspect, the invention provides a vector comprising saidpolynucleotide. The vector may be any vector capable of transferring DNAto a cell. Preferably, the vector is an integrating vector or anepisomal vector.

Preferred integrating vectors include recombinant retroviral vectors. Arecombinant retroviral vector will include DNA of at least a portion ofa retroviral genome which portion is capable of infecting the targetcells. The term “infection” is used to mean the process by which a virustransfers genetic material to its host or target cell. Preferably, theretrovirus used in the construction of a vector of the invention is alsorendered replication-defective to remove the effect of viral replicationon the target cells. In such cases, the replication-defective viralgenome can be packaged by a helper virus in accordance with conventionaltechniques. Generally, any retrovirus meeting the above criteria ofinfectivity and capability of functional gene transfer can be employedin the practice of the invention. Lentiviral vectors are especiallypreferred.

Suitable retroviral vectors include but are not limited to pLJ, pZip,pWe and pEM, well known to those of skill in the art. Suitable packagingvirus lines for replication-defective retroviruses include, for example,ΨCrip, ΨCre, Ψ2 and ΨAm.

Other vectors useful in the present invention include adenovirus,adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirusvectors. A preferred episomal vector is the adenovirus. Adenovirusvectors are well known to those skilled in the art and have been used todeliver genes to numerous cell types, including airway epithelium,skeletal muscle, liver, brain and skin (Hitt et al, 1997; Anderson,1998).

A further preferred vector is the adeno-associated (AAV) vector. MVvectors are well known to those skilled in the art and have been used tostably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletalmuscle, brain, erythroid and haematopoietic stem cells for gene therapyapplications (Philip et al., 1994; Russell et al., 1994; Flotte et al.,1993; Walsh et al., 1994; Miller et al., 1994; Emerson, 1996).International Patent Application WO 91/18088 describes specific AAVbased vectors.

Other preferred episomal vectors include transient non-replicatingepisomal vectors and self-replicating episomal vectors with functionsderived from viral origins of replication such as those from EBV, humanpapovavirus (BK) and BPV-1. Such integrating and episomal vectors arewell known to those skilled in the art and are fully described in thebody of literature well known to those skilled in the art. Inparticular, suitable episomal vectors are described in WO98/07876.

Mammalian artificial chromosomes can also be used as vectors in thepresent invention. The use of mammalian artificial chromosomes isdiscussed by Calos (1996).

In a further preferred embodiment, the vector of the present inventionis a plasmid. The plasmid may be a non-replicating, non-integratingplasmid.

The term “plasmid” as used herein refers to any nucleic acid encoding anexpressible gene and includes linear or circular nucleic acids anddouble or single stranded nucleic acids. The nucleic acid can be DNA orRNA and may comprise modified nucleotides or ribonucleotides, and may bechemically modified by such means as methylation or the inclusion ofprotecting groups or cap- or tail structures.

A non-replicating, non-integrating plasmid is a nucleic acid which whentransfected into a host cell does not replicate and does notspecifically integrate into the host cell's genome (i.e. does notintegrate at high frequencies and does not integrate at specific sites).

Replicating plasmids can be identified using standard assays includingthe standard replication assay of Ustav and Stenlund (1991).

The present invention also provides a host cell transfected with theisolated polynucleotide or vector comprising such a polynucleotide ofthe present invention. The host cell may be any eukaryotic cell.Preferably it is a mammalian cell. More preferably, it is a human celland, most preferably, it is an autologous cell derived from the patientand transfected or transduced either in vivo or ex vivo.

Numerous techniques are known and are useful according to the inventionfor delivering the vectors described herein to cells, including the useof nucleic acid condensing agents, electroporation, complexing withasbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationicliposomes, lipopolyamines, polyornithine, particle bombardment anddirect microinjection (reviewed by Kucherlapati and Skoultchi, 1984;Keown et al., 1990; Weir, 1999; Nishikawa and Huang, 2001).

A vector of the invention may be delivered to a host cellnon-specifically or specifically (i.e., to a designated subset of hostcells) via a viral or non-viral means of delivery. Preferred deliverymethods of viral origin include viral particle-producing packaging celllines as transfection recipients for the vector of the present inventioninto which viral packaging signals have been engineered, such as thoseof adenovirus, herpes viruses and papovaviruses. Preferred non-viralbased gene delivery means and methods may also be used in the inventionand include direct naked nucleic acid injection, nucleic acid condensingpeptides and non-peptides, cationic liposomes and encapsulation inliposomes.

The direct delivery of vector into tissue has been described and some,mostly short-term, gene expression has been achieved. Direct delivery ofvector into thyroid (Sikes et al., 1994) melanoma (Vile et al., 1993),skin (Hengge et al., 1995), liver (Hickman et al., 1994) and afterexposure of airway epithelium (Meyer et al., 1995) is clearly describedin the prior art. Direct DNA injection into muscle has been shown togive longer-term expression (Wolff et al., 1990).

Various peptides derived from the amino acid sequences of viral envelopeproteins have been used in gene transfer when co-administered withpolylysine DNA complexes (Plank et al., 1994; Trubetskoy et al., 1992;WO 91/17773; WO 92/19287) and Mack et al., (1994) suggest thatco-condensation of polylysine conjugates with cationic lipids can leadto improvement in gene transfer efficiency. International PatentApplication WO 95/02698 discloses the use of viral components to attemptto increase the efficiency of cationic lipid gene transfer.

Nucleic acid condensing agents useful in the invention include spermine,spermine derivatives, histones, cationic peptides, cationic non-peptidessuch as polyethyleneimine (PEI) and polylysine. ‘Spermine derivatives’refers to analogues and derivatives of spermine and include compounds asset forth in International Patent Application WO 93/18759 (publishedSep. 30, 1993).

Disulphide bonds have been used to link the peptidic components of adelivery vehicle (Cotten et al., 1992); see also Trubetskoy et al.(supra).

Delivery vehicles for delivery of DNA constructs to cells are known inthe art and include DNA/poly-cation complexes which are specific for acell surface receptor, as described in, for example, Wu and Wu, 1988;Wilson et al., 1992; and U.S. Pat. No. 5,166,320.

Delivery of a vector according to the invention is contemplated usingnucleic acid condensing peptides. Nucleic acid condensing peptides,which are particularly useful for condensing the vector and deliveringthe vector to a cell, are described in International Patent ApplicationWO 96/41606. Functional groups may be bound to peptides useful fordelivery of a vector according to the invention, as described in WO96/41606. These functional groups may include a ligand that targets aspecific cell-type such as a monoclonal antibody, insulin, transferrin,asialoglycoprotein, or a sugar. The ligand thus may target cells in anon-specific manner or in a specific manner that is restricted withrespect to cell type.

The functional groups also may comprise a lipid, such as palmitoyl,oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethyleneglycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such asthe HA peptide of influenza virus; or a recombinase or an integrase. Thefunctional group also may comprise an intracellular trafficking proteinsuch as a nuclear localisation sequence (NLS), an endosome escape signalsuch as a membrane disruptive peptide, or a signal directing a proteindirectly to the cytoplasm.

The invention provides a pharmaceutical composition comprising theisolated polynucleotide, vector or host cell of the invention asdescribed, and a pharmaceutically acceptable excipient, carrier, diluentor buffer.

In another aspect, the invention provides a product comprising acombination of the isolated polynucleotide, vector or host cell of theinvention as described, and a prodrug capable of being converted into anactive cytotoxic compound by the enzyme encoded by said nucleotide orvector, or expressed by the host cell, as a combined medicament forsimultaneous, separate or sequential use in the treatment of asepticloosening of orthopaedic implants, such as prostheses used for total hiparthroplasty. The loosening may be of the acetabular component or thefemoral component, or both. The invention is not restricted toprostheses of the hip, but may be applied to any intraosseous implantwhere aseptic loosening may occur. Accordingly its use for prosthesesused in arthroplasty of the knee, elbow, shoulder, or any other joint ofthe skeleton is specifically envisaged.

Such use need not be restricted to human use. The method is equallyapplicable to loosening of prostheses of animal joints, in particularhorses and dogs.

Preferably, the enzyme of such a product is a nitroreductase, morepreferably a nitroreductase suitable for activation of CB1954. Mostpreferably, the prodrug is CB1954.

Alternatively, the enzyme is a cytochrome P450 of a type hereindescribed. Most preferably the prodrug is acetaminophen.

In a further aspect of the invention, the use of a product comprising acombination of at least one vector, which comprises an isolatedpolynucleotide encoding an enzyme capable of converting a prodrug intoan active cytotoxic compound, expression of the enzyme being controlledby an operably-linked promoter; and a prodrug capable of being convertedinto an active cytotoxic compound by said enzyme, for the manufacture ofa combined medicament for simultaneous, separate or sequential use inthe treatment of aseptic loosening of orthopaedic implants is provided.

The promoter controlling expression of the prodrug-converting enzyme maybe a non-cell type specific promoter. Preferably, said promoter giveshigh levels of expression in a variety of tissues and cell types. Morepreferably it is selected from at least one of the following; the CMVimmediate/early promoter, RSV LTR), murine leukaemia virus LTR, SV40early or late promoters, HSV tk promoter. In a further preferredembodiment it is the human cytomegalovirus immediate/early promoter.Alternatively, it is the mouse cytomegalovirus immediate/early promoter.

In an alternative preferred product for use in the manufacture of acombined medicament for simultaneous, separate or sequential use in thetreatment of aseptic loosening of orthopaedic implants, expression ofthe enzyme is controlled by an operably-linked promoter, which providessubstantially cell-type specific expression.

More preferably expression is restricted to cells of themonocyte/macrophage lineage or fibroblasts, in which case the promotermay be naturally linked to a gene selectively expressed in cells of oneof these lineages, as described above.

Most preferably expression is restricted to osteoclasts or osteoclastprecursors, as described above.

Preferably, the enzyme is a nitroreductase, and most preferably anitroreductase suitable for activating CB1954. In this case it ispreferred that the prodrug is CB1954.

Alternatively, the enzyme may be a cytochrome P450 as herein described.In this case it is preferred that the prodrug is acetaminophen.Alternatively, it may be a conventional cytotoxic, especiallycyclophosphamide or ifosfamide.

A further aspect of the invention provides a method of treating asepticloosening of orthopaedic implants comprising administering to a patienta vector encoding an enzyme capable of converting a prodrug into anactive cytotoxic compound, allowing the expression of said enzyme intarget cells, and administering a suitable prodrug.

As will be appreciated by those of skill in the art, dosages aredetermined by clearly understood clinical parameters. However, it ispreferred that the viral dose per joint treated is between 10⁵ and 10¹²pfu, more preferably between 10⁶ and 10¹² pfu, further preferablybetween 10⁷ and 10¹² pfu and most preferably between 10⁹ and 10¹² pfu.Similarly, the dose of prodrug is dependent on clinical parameters. Inthe case of CB1954, it is preferred that the dose should be between 5and 40 mg m⁻², preferably between 5 and 30 mg m⁻², further preferablybetween 10 and 25 mg m⁻², more preferably between 15 and 25 mg m⁻², andmost preferably 24 mg m⁻² given by intra-articular injection.

It is preferred that viral vectors are not co-administered with aniodine-containing contrast medium, since such media can inhibit viraltransduction of target cells. Where the injection is to be directed bywith arthroscopic visualisation, it is preferred that an air arthrogramis performed, or a contrast medium that does not inhibit viraltransduction is used.

Preferably, the vector is administered by intra-articular orperiprosthetic injection.

It is also preferred that the prodrug is administered by intra-articularor periprosthetic injection. Alternatively, the prodrug may beadministered systemically, more preferably parenterally. However someprodrugs, particularly acetaminophen, may be administered orally.

In one preferred embodiment, expression of the prodrug-converting enzymeis controlled by a promoter that provides non-cell type specificexpression. In this case expression is not restricted to a particulartissue or cell type. As described herein, it is preferred that suchpromoters give high levels of expression in a variety of cell types.Examples of suitable promoters include the cytomegalovirusimmediate/early promoter, Rous sarcoma virus long terminal repeat (RSVLTR), murine leukaemia virus LTR, simian virus 40 (SV40) early or latepromoters, herpes simplex virus (HSV) thymidine kinase (tk) promoter

In an alternative preferred embodiment, expression of the prodrugconverting enzyme is controlled by a promoter that providessubstantially cell-type specific expression. Preferably, this issubstantially restricted to cells of the monocyte/macrophage lineage.Suitable promoters are described herein. Alternatively, it is restrictedto expression in fibroblasts. More preferably, it is substantiallyrestricted to osteoclasts or osteoclast precursors. Suitable andpreferred promoters include the TRAP, RANK, and cathepsin K promoters.

As herein described preferred prodrug converting enzymes includenitroreductases, particularly those suitable for activating CB1954, andcytochrome P450 enzymes, particularly those most suitable for activatingacetaminophen to NABQI. Preferred prodrugs accordingly include CB1954and acetaminophen. However, in the case of cytochrome P450 enzymes,conventional cytotoxic prodrugs such as cyclophosphamide are alsosuitable.

In a further aspect of the invention, an isolated polynucleotide, orvector comprising such a polynucleotide or host cell comprising either,may encode, or express, a protein or peptide that is directly toxic tocells. In this case, no prodrug administration is required. Because ofthe self-contained nature of the joint/periprosthetic space surroundedby the interface tissue, it is possible to introduce vectors into thispathological space so that cells therein are transfected or transduced,causing them to express toxic products. Among the toxins that could beencoded and used in this way are ricin, abrin, diphtheria toxin,Pseudomonas exotoxin, DNase, RNase and botulinum toxin.

Preferably, the expression of such directly toxic molecules is under thecontrol of a promoter providing substantially cell-type specificexpression as herein described. In this way, expression of the toxin isrestricted to target cells defined both by the physical constraints ofthe space into which the vector is introduced and the phenotype of thecells transfected or transduced. In this way, fibroblasts, orinflammatory cells such as activated cells of the monocyte/macrophagelineage, or specific cells such as osteoclasts and their precursorsdirectly responsible for bone resorption, are targeted.

Accordingly, an isolated polynucleotide encoding a toxic peptide orprotein is provided, wherein expression of the toxin is controlled by apromoter providing substantially cell-type specific expression.Preferably, this expression is restricted to cells of themonocyte/macrophage lineage. Alternatively, expression is restricted tofibroblasts. More preferably, expression is restricted to osteoclastsand osteoclast precursor cells. As described herein, suitable andpreferred promoters include the c-fes and CD68 promoters to providemacrophage-specific expression and the TRAP, RANK and cathepsin Kpromoters to provide osteoclast-specific expression. Suitable andpreferred toxins encoded include ricin, abrin, diphtheria toxin,Pseudomonas exotoxin, DNase, RNase and botulinum toxin.

Also provided is a vector comprising said polynucleotide and a host cellcomprising either, and a pharmaceutical composition comprising anisolated polynucleotide or a vector as herein described, and apharmaceutically acceptable excipient, carrier, diluent or buffer.

In a further embodiment is provided a product comprising an isolatedpolynucleotide, vector or host cell encoding or expressing a toxicpeptide or protein as herein described, as a medication for thetreatment of aseptic loosening of orthopaedic implants. Said expressionmay be under the control of a non-cell type specific promoter givinghigh levels of expression in cells of a variety of types. Preferably,said expression is controlled by a promoter providing substantiallycell-type specific expression as herein described.

Also provided is the use of such products in the manufacture of amedicament for the treatment of aseptic loosening of orthopaedicimplants.

In a further aspect is provided a kit for treatment of aseptic looseningof orthopaedic implants comprising:

-   -   a) An isolated polynucleotide or vector encoding an enzyme        capable of converting a prodrug into an active cytotoxic        compound, expression of which enzyme being controlled by an        operably-linked promoter, in a pharmaceutically acceptable        buffer;    -   b) A prodrug capable of being converted into an active cytotoxic        compound by said enzyme, in a pharmaceutically acceptable        buffer;    -   c) An tissue-digesting solution comprising at least one enzyme        selected from the list consisting of collagenase, elastase,        hyaluronidase, in a pharmaceutically acceptable buffer; and/or a        chelator such as EDTA, EGTA etc.    -   d) A cement suitable for the refixation of said orthopaedic        implant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts aseptic loosening of a hip prosthesis. A is a radiographof loosened prosthesis in situ. B is an arthrogram of a hip joint with aloosened prosthesis. The contrast medium is injected into the jointspace under fluoroscopic guidance. The picture shows that a part of thearea around the prosthesis (periprosthetic space) is filled withcontrast medium. This proves that the prosthesis is loose in that area.C shows a schematic representation of a hip joint with a loosenedprosthesis. The gray area indicates the joint space, which is continuouswith the periprosthetic space. When injecting a fluid into the jointspace, this will spread through the area which is marked gray in theimage.

FIG. 2 shows the killing effect of infection withnitroreductase-encoding adenoviral vectors and subsequent exposure tothe prodrug CB1954 at the concentrations shown on interface cells fromtissue taken from two revision surgery patients as described in Example3. FIG. 2 a shows data from patient LI003 P3 and FIG. 2 b shows datafrom patient LI002 P4.

FIG. 3 shows the results of X-Gal staining of samples of intactinterface tissue taken from patient L1014 infected with various doses ofa Lac Z-encoding adenoviral vector, as described in Example 4.

The numbered wells contain tissue treated as follows:

1. Noninfected interface tissue2. Interface tissue+3.6×10⁴ pfu Ad.CMV.LacZ3. Interface tissue+3.6×10⁵ pfu Ad.CMV.LacZ4. Interface tissue+3.6×10⁶ pfu Ad.CMV.LacZ5. Interface tissue+3.6×10⁷ pfu Ad.CMV.LacZ6. Interface tissue+3.6×10⁸ pfu Ad.CMV.LacZ7. Interface tissue+3.6×10⁹ pfu Ad.CMV.LacZ

FIG. 4 shows transduction of interface cells following incubation withsix different concentrations of Ad.CMV.LacZ (0, 25, 50, 100, 200 and 400pfu/cell). After three days, cells were fixed and stained with X-galreaction mix. The percentage of transduced (blue) cells was counted. Thefigure shows the means and standard deviations of 12 independentexperiments.

FIG. 5 shows the lack of toxicity of iotrolan (Isovist) contrast mediumon interface cells. Interface cells were exposed to contrast medium(iotrolan) for 4 hours. After 3 days of cell culturing viability of thecells was measured (n=12).

FIG. 6 shows the effect of iotrolan on HAdV5-transduction of interfacecells. Cells were exposed to different concentrations of Ad.CMV.LacZ:((▴) 0 pfu/cell, (▪) 25 pfu/cell; () 100 pfu/cell; (♦) 200 pfu/cell.(n=4)) and contrast medium for four hours, after which the cells werefixed and stained with X-gal. Percentage of transduced cells wasdetermined by counting blue cells.

FIG. 7 shows pre-(A) and post-injection (B) images from Patient 1showing an increased cement mass in the greater trochanteric region.

FIG. 8 shows pre-(A) and post-injection (B) images from Patient 2.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are meant to illustrate the invention and do notlimit it in any way. Persons of ordinary skill in the art will recognizemodifications within the spirit and scope of the invention as set forthin the appended claims.

Example 1 Procedure for Treatment with CTL102(Ad5-NTR and CB1954)Materials

The drug product, CTL102 injection, is a sterile, clear or virtuallyclear, aqueous liquid solution containing CTL102 virions at a nominalmean potency of 2×10¹¹ particles ml⁻¹, buffered at pH 7.4.

CB1954 is formulated as a sterile solution in solvent(N-methylpyrrolidone: polyethylene glycol, 2:7 v/v with 17.8 mg CB1954ml⁻¹). Just prior to use, the prodrug in solvent is diluted in sterilesaline to a maximum final CB1954 concentration of 5 mg ml⁻¹.

To stabilise the prosthesis, low viscosity bone cement (Simplex® P withtobramycin from Howmedica Inc, Rutherford, N.J., USA) is used. Thisradiopaque bone cement is a mixture of a liquid monomer component (2 ml97.4% methylmethacrylate, 2.6% N,N-dimethyl-p-toluidine, 75 ppmhydroquinone) and a polymer powder (6 g polymethylmethacrylate, 30 gmethylmethacrylate-styrene copolymer, 4 g barium sulphate, 1 gtobramycin sulphate). The components are vacuum mixed (0.9 bar, 1minute) immediately before use.

For arthrography, Hexabrix 320 (ioxaglate sodium meglumine, Guerbet,Roissy Charles de Gaulle Cedex, France) contrast medium is used.

Procedure

Following careful flushing of the joint to remove synovial fluid andinflammatory exudate that may contain neutralising anti-adenovirusantibodies, 3×10⁹ pfu CTL102 is injected intra-articularly resulting indelivery of vector to cells throughout the periprosthetic space. After48 hours, to allow transduction of target cells and expression of thenitroreductase transgene, CB1954 (at a dosage of 24 mg m⁻²) is injectedintra-articularly. To assure free access of CTL 102 and CB 1954 to theperiprosthetic space it is preferred that patients are selected who havean arthrogram that shows contrast medium around the prosthesis. It islikely, therefore, that patients will usually undergo threearthrographies (one to assure access of contrast medium, one to injectthe viral vector, and one to inject the CB 1954 prodrug).

In some circumstances after a number of days dead interface tissue maybe removed by flushing or physical debridement, as appropriate. When theinterface tissue is successfully diminished the prosthesis is refixated.To re-anchor the prosthesis to the bone, cement is injected in theperiprosthetic space. For the flushing of the periprosthetic space andinjection of the cement a number of holes are drilled through the boneinto the periprosthetic space. This depends on the design of theprosthesis used. In many common designs, four is the minimum, becausethree holes are necessary for the femoral component to fixate in 3Dspace and one is necessary to fixate the acetabulum. As the bonebiopsies are rather painful and the bone cannot be anaesthetisedlocally, these procedures are performed under general or spinalanaesthesia.

Example 2 Production of CTL102 (Ad5-NTR) Materials and Methods

CTL102 was constructed as described in Djeha et al (2001) by homologousrecombination in PerC6 helper cells. The cells were transfected at 90%confluence with an equimolar mixture of the transfer vector pTX0375 andthe backbone vector pPS1160 complexed with Lipofectamine transfectionreagent (Life Technologies).

pTX0375 was constructed in two stages: (i) the CMV promoter/enhancerfused to the NTR gene was excised from pTX0340 as a 1.5-kb BamHI-partialBgIII fragment and cloned into the unique BamHI site of pSW107, which isa pBluescript-based vector (Stratagene) that contains the human b-globinIVS II fused to the human complement 2 gene polyadenylation sequenceadjacent to the BamHI site. A plasmid, pTX0374, which contains theCMV.NTR fragment in the required orientation, was identified by PCRusing the T3 primer (5′-ATTAACCCTCAC-TAAAG-3′) which anneals to the CMVpromoter/enhancer, and an NTR primer, ECN2 (5′-TCTGCTCGGCCTGTTCC-3′).(ii) The complete NTR expression cassette was excised from pTX0374 as a2.5-kb SpeI fragment and cloned into the unique SpeI site of theE1-deleted adenovirus transfer vector pPS1128 in a left-to-rightorientation with respect to Ad5 sequences. pPS1128 is a pUC19-basedplasmid that contains Ad5 sequences from the left-hand ITR tonucleotides (nt.) 359 fused to NT 3525-10589.

pPS1160 was constructed by PacI linearisation of pPS1128, ligation witha PacI-compatible adaptor (5′-TACATCTAGATAAT-3′+5′-P-TTATCTAGAT-GTA-3′)containing an XbaI site, followed by XbaI digestion to release a 7-kbXbaI fragment containing Ad5 sequences 3524-10589. This was then clonedinto XbaI-linearised pPS1022, a pUC19-based plasmid containing Ad5sequences from nt. 10589 to the right-hand ITR but lacking NT 28592 to30470 (E3 region). Recombinants containing the fragment in the requiredorientation were identified by PCR using primers flanking the XbaI siteat 10589 (rightward, 5′-TCGAGTCAAATACGTAGTCGT-3′; leftward,5′-TGTTTCCGGAGGAATTTGCAA-3′). A plasmid, pPS1160/18, was confirmed tocontain a single copy of the XbaI fragment (pPS1160/18) by HindIII andPstI digestion.

Transfected PerC6 cells were harvested following the appearance ofextensive CPE (about 7-9 days after transfection) and recombinant virusreleased by three freeze-thaw cycles in infection medium (DMEM, 1% FCS,2 mM MgCl₂). After two rounds of plaque purification on PerC6 cells theviruses were grown to large scale and purified by CsCl densitycentrifugation. Banded virus was dialysed against an excess of storagebuffer (10 mM Tris, pH 7.4, 140 mM NaCl, 5 mM KCl, 0.6 mM Na₂ HPO₄, 0.9mM CaCl₂, 0.5 mM MgCl₂, and 5% sucrose), snap-frozen in aliquots inliquid nitrogen, and stored at −280° C. Particle concentrations weredetermined using the BCA Protein Assay Reagent (Pierce, Rockford, Ill.)and the conversion factor 1 mg/ml=3.4×10¹² virus particles/ml.Infectious titres were determined by plaque assay. Genomic DNA wasisolated from banded adenovirus by digestion with proteinase K/SDS,phenol-chloroform extraction, and ethanol precipitation andcharacterised by restriction digestion.

Example 3 Killing of Interface Tissue from Patients with CTL102 andCB1954

In order to demonstrate the feasibility of using a virally deliveredenzyme-prodrug system to kill interface cells, cells taken from twopatients during revision surgery were cultured in vitro, incubated withCTL102 at a range of MOIs and subsequently exposed to CB1954. Cellviability was then determined using a metabolic activity assay.

Method Interface Tissue Samples

For all experiments described, interface cells were used. Interfacetissue was removed from the periprosthetic space during revision-surgeryby an orthopedic surgeon and collected in sterile phosphate bufferedsaline (PBS). Connective tissue and fat were removed thoroughly and theinterface tissue was digested for at least two hours at 37° C. usingcollagenase 1A (1 mg/ml; Sigma, St Louis, Mo., USA). Cells were thenharvested by filtering the tissue/collagenase substance through a 200 μmfilter (NPBI, Emmer-Compascuum, The Netherlands). The cells werecultured in 75 cm² flasks (Celistar, Greiner, Alphen aan de Rijn, TheNetherlands) with Iscove's modified Dulbecco's medium (IMDM;Biowitthaker, Verviers, Belgium), supplemented with glutamax (GibcoBRL,Paisley, UK), penicillin and streptomycin (Boehringer Mannheim,Germany), and 10% fetal calf serum (FCS; GibcoBRL, Paisley, UK) at 37°C. and 5% CO₂.

Before each experiment interface cells were detached from the flasksusing 0.25% trypsin (GibcoBRL, Paisley, UK). The cells were counted in abürker counter and death cells were excluded by trypan blue. Cells wereseeded in a 96 wells-plate (flat bottom) at a density of 5,000 cells perwell. Cells were incubated overnight to allow attachment to the bottom.Before each experiment the wells were washed twice with IMDM. For theexperiments passage 2 to 4 interface cells were used. Light microscopyindicated that more than 95% of the cells were interface cells.

Transduction and Cell Killing Assay Protocol

Day 0: Interface cells from 2 patients were seeded at 5000 cells/well inIMDM (10% FCS) in 96 wells plates, 100 μl per well.

Day 1: Cells were infected with CTL102 (or diluent) at 0, 1, 5, 25, 100,200 IU/cell in IMDM (10% FCS), 50 μl per well.

Day 2: Cells were washed twice with in IMDM (10% FCS), hereafter cellswere incubated for 2 hr or 24 hr with CB1954 (or vehicle) at 0, 0.1,0.5, 1, 5 and 50 μM in IMDM (10% FCS, 10% HS), 50 μl per well.

Day 2/3: Cells were washed once with IMDM (10% FCS) and then incubatedin IMDM (10% FCS, 10% HS), 5 μl per well.

Day 4: Photographs were taken. Medium was refreshed with IMDM (10% FCS),10 μl WST reagent (Roche) was added and the plates were incubated for 2hr. Hereafter the absorbance at 415 nm was measured.

Results

As shown in FIGS. 2A and 2B, virus and CB1954-dose dependent killing wasobserved for cells from both patients. Importantly, efficient (90%)killing was observed with virus and CB1954 doses (200 virus pfu/cell anda CB1954 concentration of 50 μM) that is readily achievable in theclinic.

These results demonstrate that interface cells can be transduced by anHAdV-5-vector and killed by the NTR/CB1954 approach. Human adenovirus 5is capable of infecting a broad range of dividing and non-dividing humancells including fibroblasts and macrophages (Djeha et al, 2001).

Killing of cells by GDEPT has been studied before in various cell lines,using various approaches. The NTR/CB1954 approach is attractive forclinical evaluation for several reasons: (1) it generates a toxic agentthat can kill both dividing and non-dividing cells, (2) induction ofcell death occurs by a p53-independent mechanism, and (3) CB1954 iswell-tolerated in man (Djeha et al, 2001). Cell killing by theNTR/CB1954 approach has been proved effective in a variety of humancancer cells (Chung-Faye et al, 2001; Bilsland et al, 2003, Green et al,2003; McNeish et al, 1998; Shibata et al, 2002; Weedon et al, 2000;Wilson et al, 2002), but has not previously been studied in synovial orinterface cells. The current study shows that interface cells can beeffectively killed by the NTR/CB1954 approach.

For the current study passage 2 to 4 interface cells were used. Thesepassages were used to maximally reduce culture artefacts. On the onehand, in very low passages (0 and 1) there is a risk for presence ofcontaminating cells (especially macrophages), which decreases withhigher passages. On the other hand, at higher passages the risk ofsubstantial in vitro alteration/growth selection exists (especially atpassages higher than 4) (Zimmerman et al, 2001). In the current study,cultured interface cells of different patients were used. For theinterpretation of the results the data of all patients were pooled.However, it must be noted that individual differences in transducibilitywere observed.

Example 4 Efficient Infection of Intact Interface Tissue with AdenovirusVectors

The experiment outlined in Example 3 confirmed that cultured interfacecells are Ad5-infectable. However, when a cell is present within anintact tissue, access of the virus to the cell surface may be prevented,for instance by the extracellular matrix and by the low rate of virusdiffusion through the extracellular space. In view of this, theinfectability of fresh intact interface tissue was examined using aLacZ-expressing adenovirus and Xgal staining of LacZ-expressing tissue.Using this approach, a virus dose-dependent increase in gene expressionwas observed, with strong levels of gene expression with the two highestvirus doses tested (FIG. 3).

Method

Interface tissue (LI014) was obtained from a revision operation of thehip of a rheumatoid arthritis patient. The tissue was cut in 7 piecesand the pieces were put in 10 ml round bottom tubes. Differentconcentrations of Ad.CMV.LacZ (0, 3.6×10⁴, 3.6×10⁵, 3.6×10⁶, 3.6×10⁷,3.6×10⁸, 3.6×10⁹ pfu) in 200 μl IMDM/10% FCS were added. The tissueswere incubated at 37° C. for 2 hours, the tubes were shaken every 10 to15 minutes. Hereafter 5 ml IMDM/10% FCS was added and after an overnightincubation the tissues were rinsed 3× with PBS and subsequently put in 5ml Xgal colouring solution and incubated for 3.5 hours at 37° C. Thetissues were rinsed 3× with PBS and fixed in 10% formalin.

Results

The tissues with the highest added amounts of Ad.CMV.LacZ have areas ofdark blue staining, which is evident down to an infection at 3.6×10⁷ pfuAd.CMV.LacZ. Demonstrating that infection of cells in intact interfacetissue is effective.

Embedded paraffin sections of the tissues were examined microscopicallyand the presence of stained, infected cells was confirmed.

Example 5 Transduction of Interface Tissue and Effect of Contrast Medium

To test further the susceptibility of interface cells to humanadenovirus 5 (HAdV-5)-based vectors, primary cultures of interface cellswere exposed to the HAdV-5 vector Ad.CMV.LacZ. Twenty-four hourspost-infection the cells were stained with X-gal solution forβ-galactosidase reporter gene expression. The transduction efficiencyincreased with increasing vector concentration. At 400 plaque formingunits/cell the percentage of cells expressing the reporter gene was 88%(sd 4.0) (FIG. 4). Thus HAdV-5 vectors can transduce interface cells.

Materials and Methods Adenoviral Vectors

The Ad.CMV.LacZ (van der Eb et al, 2002) vector is identical to CTL102,but the E. coli lacZ gene replaces the ntr gene.

Transduction Assays

To study the transducibility of interface cells by HAdV-5, interfacecells were infected with Ad.CMV.LacZ vector (in concentrations of 0, 25,50, 100, 200, 400 pfu/cell). Twenty-four hours post infection the cellswere washed twice with IMDM, and cultured for two days. Medium wasrefreshed each day. On day three, the monolayer cultures were washedtwice with PBS and fixed with 0.2% glutaraldehyde and 2% formaldehyde inPBS for 10 minutes at 4° C. Subsequently cells were washed twice withPBS and stained for β-galactosidase activity in 50 μl of reaction mix (1mg/ml X-gal (Eurogentec, Seraing, Belgium), 5 mM potassium ferrocyanide,5 mM potassium ferricyanide, 2 mM MgCl₂ in PBS) for 2 hours at 37° C.The percentage of transduced cells was assessed by counting at least 100interface cells, using light microscopy. All conditions were tested induplicate.

Effect of Contrast Medium on Interface Cells

Interface cells were seeded in 96-wells plates. Into each well 50 μl ofIMDM/20% FCS and 50 μl of a solution containing contrast medium and 0.9%NaCl in various concentrations (0, 12.5, 25, and 50% contrast medium)were added. The contrast medium used was the low-osmolarity, nonionicdimer iotrolan (Isovist; Schering, Berlin, Germany). After four hours ofexposure to the contrast medium, the cells were washed twice andincubated in IMDM/10% FCS. The cells were cultured for three more days,changing the culture medium every day. On day four, cell viability wasdetermined with the WST-1 cell viability assay kit (Roche, Mannheim,Germany) according to the manufacturers protocol.

Effect of Contrast Medium on HAdV-5-Transduction of Interface Cells

Interface cells were seeded in 96-wells plates. After overnightincubation cells were infected with Ad.CMV.LacZ (concentrations of 0,25, 100, and 200 pfu/cell) in IMDM/20% FCS, 50 μl per well. Fifty μlIotrolan (Isovist) in 0.9% NaCl was added in concentrations of 0, 25,50, and 100%. (When diluted in the culture medium these concentrationsdecreased to 0, 12.5, 25, and 50%.) Four hours after infection, thecells were washed twice with IMDM and incubated for the rest of the dayin IMDM/10% FCS at 37° C. and 5% CO₂. The Ad.CMV.LacZ transduced cellswere cultured for three days after removal of the vector and contrastmedium. Subsequently, the cells were fixed and stained forβ-galactasidase activity. The transduction rate was assessed asdescribed above.

Statistical Analysis

A univariate analysis of variance and Spearman's correlation was used tostudy the interaction between vector and prodrug and between vector andcontrast medium and to study the effect of CB1954 on viability of thecells. A Mann-Whitney test for independent groups was performed todetermine the difference in cell killing between the cells that wereexposed to contrast medium and the non-exposed cells. In the experimentto study the effect of transient exposure to contrast medium ontransduction of HAdV-5-vector Spearman's correlation between contacttime and viability and between delay time and viability was tested. Forall statistical analyses p<0.05 was the level of statisticalsignificance.

Results Effect of Contrast Medium on Interface Cells

The toxicity of contrast medium (iotrolan) on interface cells wasevaluated (FIG. 5). Iotrolan does not affect the viability of the cellsat any concentration (p=0.563).

Adding of contrast medium to the interface cells for four hours does notlead to killing of the cells.

Effect of Contrast Medium on HAdV-5 Transduction of Interface Cells

The effect of contrast medium (iotrolan) on HAdV5-transduction ofinterface cells was investigated with Ad.CMV.LacZ. Transducibility ofthe cells increases with the concentration of HAdV-5 vector. However,the contrast medium has restraining influence on the transductionefficiency. With higher concentrations of iotrolan, the HAdV-5 vectorconcentration has less effect on gene transfer efficiency. At a contrastmedium concentration of 50% none of the cells were transduced (FIG. 6).The effect of iotrolan on the transduction is statistically significant(p<0.001). Furthermore, differences between cells from differentindividuals (n=6) have been observed. To evaluate the effect of contrastmedium on cell killing by NTR/CB1954, the previously describedexperiment for the efficiency of cell killing was repeated in thepresence of contrast medium. The results showed that, in the presence ofcontrast medium, cells are not killed by the NTR/CB1954 approach(results not shown). The presence of Hexabrix 320 contrast medium alsoinhibited viral transduction (data not shown). In summary, the resultsfrom these experiments demonstrate the incompatibility of viraladministration in combination with the administration of two commonlyused contrast media. This incompatibility may be due to the presence ofiodine within the contrast media. Screening of all available contrastmedia may allow determination of a contrast medium compatible with viraltransduction.

The influence of transient exposure to contrast medium on thetransduction of interface cells was investigated. Interface cells wereexposed to contrast medium for 0 to 120 minutes and the period betweenwashing away of the contrast medium and performing the NTR/CB1954 cellkilling approach was varied. Cell killing was not correlated withcontact time (corr −0.033, p=0.691) or length of period between washingaway of the contrast medium and addition of the vector (corr −0.004,p=0.962). Killing of cells not exposed to contrast medium and thosetransiently exposed was equivalent.

Discussion

In this study the influence of contrast medium on cell killing byNTR/CB1954 was investigated in view of future clinical studies. Resultsshow that the contrast medium does not seem to have any influence on theinterface cells. However, transduction of the cells by an adenoviralvector, in the presence of contrast medium, is almost negligible. Theadenoviral vector is inactivated by the presence of contrast medium. Ina putative clinical study the viral vector will be injected in the jointspace. Normally, contrast medium is used to verify the position of theneedle in the joint. The results of this study however show that the useof contrast medium in combination with a viral vector is dissuaded.Thus, for a clinical study, we propose that alternative methods for thevisualization of the needle should be employed such as injection of airto create an “air-arthrogram”.

In conclusion, this example shows that interface cells can be killed bythe NTR/CB1954 enzyme prodrug approach.

Example 6 Clinical Outcomes

Data are available from the first two patients from a phase-1 study of12 patients with a loosened hip experiencing debilitating pain andsignificant comorbidity. On day 1 the vector was injected into the hipjoint and the prodrug injected on day 3, as described above. On day 10three holes were drilled in the femur and one in the acetabulum.Biopsies are taken from the periprosthetic space and low viscositycement (Osteopal, Biomet Merck, Sjöbo, Sweden) injected underfluoroscopic guidance.

Patient 1 is an 82-year old female with loosening of both hipprostheses, classified ASA IV (mortality risk 20.3%, American Society ofAnesthesiologists physical status classification, Saklad, 1941). Therewere no adverse effects from vector injection (3×10⁹ particles) and 24hours post-injection there was no detectable virus shedding. Twelvehours after prodrug injection the patient experienced nausea, (WHOgrade 1) which was known as a reaction to the prodrug. Also hip painincreased, which was anticipated as the initial therapy is intended tocause more loosening. 16 ml of cement was injected into periprostheticspace (see FIG. 7B) indicating significant destruction of interfacetissue creating a void into which cement could now be introduced. Thepatient was ambulated the day after surgery.

At two and four weeks after cement injection the patient had no pain inthe treated hip, and was still improving. The maximum walking distancehad increased from 4-5 metres to 30 metres. Subjective walking distanceassessed by the patient (0: 0 metres, 100: unlimited walking distance)increased from 4 to 66. The patient's pain score (0: no pain, 100:unbearable pain) decreased from 81 preoperatively to 2. In addition, shecould now sleep on her side without pain, which she had been unable todo for four years. In terms of perceived dependency (0: completelydependent on others, 100: completely independent) the score decreasedfrom 95 to 54.

Patient 2 is a 72 year old woman with loosening of her left hipprosthesis and an ASA classification of II (mortality risk 2.8%). Again,there was no detectable virus shedding 24 hours after vector injection.18 ml of cement was injected following a similar procedure (FIG. 8B).Four weeks post-treatment the pain score had decreased from 43 to 22(probably reflecting the presence of a post-operative haematoma,requiring 4-5 weeks to resolve). Specifically hip joint-related paindisappeared. Maximum walking distance increased from 500 to 2000 metres.By the 3 month follow-up, the haematoma had completely resolved and painscore had further decreased to 7. The patient continues to improve interms of walking performance and other activities.

The current study is the first to use in vivo intra-articular adenoviralmediated gene transfer in a clinical setting. The preliminary resultssuggest that gene therapy and cement injection for hip prosthesisrefixation is clinically feasible.

All references cited herein are hereby incorporated by reference intheir entireties.

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1. A method for reducing interface tissue causing aseptic loosening oforthopaedic implants comprising: a) administering to interface tissuecells by intra-articular or periprosthetic injection an effective amountof at least one vector comprising a polynucleotide encoding an enzymecapable of converting a prodrug into an active cytotoxic compound,expression of the enzyme being controlled by an operably-linkedpromoter; and b) administering an effective amount of a prodrug capableof being converted into an active cytotoxic compound by said enzyme, tosaid interface tissue; and c) allowing the expression of said enzyme insaid interface tissue cells, wherein interface tissue is reduced.
 2. Themethod according to claim 1, wherein the promoter provides non-cell typespecific expression.
 3. The method according to claim 2, wherein thepromoter is a cytomegalovirus promoter.
 4. The method according to claim1, wherein the promoter provides substantially cell-type specificexpression.
 5. The method according to claim 4, wherein expression issubstantially restricted to cells of the monocyte/macrophage lineage. 6.The method according to claim 5 wherein expression is substantiallyrestricted to osteoclasts and osteoclast precursor cells.
 7. The methodaccording to claim 1, wherein the promoter is naturally functionallylinked to a gene selected from the group consisting of; TRAP, RANK andcathepsin K.
 8. The method according to claim 1 wherein the enzyme is acytochrome P450.
 9. The method according to claim 8, wherein the prodrugis acetaminophen.
 10. A method of treating aseptic loosening oforthopaedic implants comprising: a) administering to interface tissuecells by intra-articular or periprosthetic injection to a human or otheranimal an effective amount of a vector comprising a polynucleotideencoding an enzyme capable of converting a prodrug into an activecytotoxic compound, expression of the enzyme being controlled by anoperably-linked promoter; b) allowing the expression of said enzyme insaid interface tissue cell's; c) administering an effective amount of aprodrug capable of being converted into a cytotoxic compound by saidenzyme to said interface tissue; and d) refixing the orthopaedicimplant.
 11. The method of claim 10 wherein the prodrug is administeredby intra-articular or periprosthetic injection.
 12. The method of claim10 wherein expression of said enzyme is controlled by a promoter thatprovides non-cell type specific expression.
 13. The method of claim 10,wherein expression of said enzyme is controlled by a promoter thatprovides substantially cell-type specific expression.
 14. The method ofclaim 13, wherein expression is substantially restricted to cells of themonocyte/macrophage lineage.
 15. The method of claim 14, whereinexpression is substantially restricted to osteoclasts or osteoclastprecursors.
 16. The method of claim 10, wherein the promoter isnaturally functionally linked to a gene selected from the groupconsisting of; TRAP, RANK and cathepsin K.
 17. The method of claim 10,wherein the enzyme is a cytochrome P450.
 18. The method of claim 17,wherein the prodrug is acetaminophen.